Mammalian genes involved in viral infection and tumor suppression

ABSTRACT

The present invention provides methods of identifying cellular genes necessary for viral growth and cellular genes that function as tumor suppressors. Thus, the present invention provides nucleic acids related to and methods of reducing or preventing viral infection or cancer. The invention also provides methods of producing substantially virus- free cell cultures and methods for screening for additional such genes.

This application is a continuation of U.S. application Ser. No.09/509,712, filed Mar. 31, 2000 which is the National Stage ofInternational Application No. PCT/US98/21276, filed Oct. 8, 1998 whichis a continuation of U.S. application No. 60/062,021, filed Oct. 7, 1997all of which are hereby incorporated by reference in their entireties.

This invention was made with partial government support under NationalInstitutes of Health Grant No. CA68283 and a grant from the Departmentof Veterans Affairs. The United States Government has certain rights inthe invention.

BACKGROUND

1. Field of the Invention

The present invention provides methods of identifying cellular genesused for viral growth or for tumor progression. Thus, the presentinvention relates to nucleic acids related to and methods of reducing orpreventing viral infection and for suppressing tumor progression. Theinvention also relates to methods for screening for additional suchgenes.

2. Background Art

Various projects have been directed toward isolating and sequencing thegenome of various animals, notably the human. However, mostmethodologies provide nucleotide sequences for which no function islinked or even suggested, thus limiting the immediate usefulness of suchdata.

The present invention, in contrast, provides methods of screening onlyfor nucleic acids that are involved in a specific process, i.e., viralinfection or tumor progression. For viral infection, the nucleic acidsisolated are useful in treatments for these processes because by thismethod only nucleic acids which are also nonessential to the cell areisolated. Such methods are highly useful, since they ascribe a functionto each isolated gene, and thus the isolated nucleic acids canimmediately be utilized in various specific methods and procedures.

For, example, the present invention provides methods of isolatingnucleic acids encoding gene products used for viral infection, butnonessential to the cell. Viral infections are significant causes ofhuman morbidity and mortality. Understanding the molecular mechanisms ofsuch infections will lead to new approaches in their treatment andcontrol.

Viruses can establish a variety of types of infection. These infectionscan be generally classified as lytic or persistent, though some lyticinfections are considered persistent. Generally, persistent infectionsfall into two categories: (1) chronic (productive) infection, i.e.,infection wherein infectious virus is present and can be recovered bytraditional biological methods and (2) latent infection, i.e., infectionwherein viral genome is present in the cell but infectious virus isgenerally not produced except during intermittent episodes ofreactivation. Persistence generally involves stages of both productiveand latent infection.

Lytic infections can also persist under conditions where only a smallfraction of the total cells are infected (smoldering (cycling)infection). The few infected cells release virus and are killed, but theprogeny virus again only infect a small number of the total cells.Examples of such smoldering infections include the persistence of lacticdehydrogenase virus in mice (Mahy, B. W. J., Br. Med. Bull. 41: 50-55(1985)) and adenovirus infection in humans (Porter, D. D. pp. 784-790 inBaron, S., ed. Medical Microbiology 2d ed. (Addison-Wesley, Menlo Park,Calif. 1985)).

Furthermore, a virus may be lytic for some cell types but not forothers. For example, evidence suggests that human immunodeficiency virus(HIV) is more lytic for T cells than for monocytes/macrophages, andtherefore can result in a productive infection of T cells that canresult in cell death, whereas HIV-infected mononuclear phagocytes mayproduce virus for considerable periods of time without cell lysis.(Klatzmann, et al. Science 225:59-62 (1984); Koyanagi, et al. Science241:1673-1675 (1988); Sattentau, et al. Cell 52:631-633 (1988)).

Traditional treatments for viral infection include pharmaceuticals aimedat specific virus derived proteins, such as HIV protease or reversetranscriptase, or recombinant (cloned) immune modulators (host derived),such as the interferons. However, the current methods have severallimitations and drawbacks which include high rates of viral mutationswhich render anti-viral pharmaceuticals ineffective. For immunemodulators, limited effectiveness, limiting side effects, a lack ofspecificity all limit the general applicability of these agents. Alsothe rate of success with current antivirals and immune-modulators hasbeen disappointing.

One aspect of the current invention focuses on isolating genes that arenot essential for cellular survival when disrupted in one or bothalleles, but which are required for virus replication. This may occurwith a dose effect, in which one allele knock-out may confer thephenotype of virus resistance for the cell. As targets for therapeuticintervention, inhibition of these cellular gene products, including:proteins, parts of proteins (modification enzymes that include, but arenot restricted to glycosylation, lipid modifiers [myriolate, etc.]),lipids, transcription elements and RNA regulatory molecules, may be lesslikely to have profound toxic side effects and virus mutation is lesslikely to overcome the ‘block’ to replicate successfully.

The present invention provides a significant improvement over previousmethods of attempted therapeutic intervention against viral infection byaddressing the cellular genes required by the virus for growth.Therefore, the present invention also provides an innovative therapeuticapproach to intervention in viral infection by providing methods totreat viruses by inhibiting the cellular genes necessary for viralinfection. Because these genes, by virtue of the means by which they areoriginally detected, are nonessential to the cell's survival at a levelof expression necessary to inhibit virus replication, these treatmentmethods can be used in a subject without serious detrimental effects tothe subject, as has been found with previous methods. The presentinvention also provides the surprising discovery that virally infectedcells are dependent upon a factor in serum to survive. Therefore, thepresent invention also provides a method for treating viral infection byinhibiting this serum survival factor. Finally, these discoveries alsoprovide a novel method for removing virally infected cells from a cellculture by removing, inhibiting or disrupting this serum survival factorin the culture so that non-infected cells selectively survive.

The selection of tumor suppressor gene(s) has become an important areain the discovery of new target for therapeutic intervention of cancer.Since the discovery that cells are restricted from promiscuous entryinto the cell cycle by specific genes that are capable of suppressing a‘transformed’ phenotype, considerable time has been invested in thediscovery of such genes. Some of these genes include the gene associatedby rhabdomyosarcoma (Rb) and the p53 (apoptosis related) encoding gene.The present invention provides a method, using gene-trapping, to selectcell lines that have a transformed phenotype from cells that are nottransformed and to isolate from these cells a gene that can suppress amalignant, or transformed, phenotype. Thus, by the nature of theisolation process, a function is associated with the isolated genes. Thecapacity to select quickly tumor suppressor genes can provide uniquetargets in the process of treating or preventing, and even fordiagnostic testing of, cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention utilizes a “gene trap” method along with aselection process to identify and isolate nucleic acids from genesassociated with a particular function. Specifically, it provides a meansof isolating cellular genes necessary for viral infection but notessential for the cell's survival, and it provides a means of isolatingcellular genes that suppress tumor progression.

The present invention also provides a core discovery that virallyinfected cells become dependent upon at least one factor present inserum for survival, whereas non-infected cells do not exhibit thisdependence. This core discovery has been utilized in the presentinvention in several ways. First, inhibition of the “serum survivalfactor” can be utilized to eradicate persistently virally infected cellsfrom populations of non-infected cells. Inhibition of this factor canalso be used to treat virus infection in a subject, as further describedherein. Additionally, inhibition of or withdrawal of the serum survivalfactor in tissue culture allows for the detection of cellular genesrequired for viral replication yet nonessential for an uninfected cellto survive. The present invention further provides several such cellulargenes, as well as methods of treating viral infections by inhibiting thefunctioning of such genes.

The invention also provides cellular genes whose overexpression isassociated with inhibition of viral growth and/or reproduction.

The present method provides several cellular genes that are necessaryfor viral growth in the cell but are not essential for the cell tosurvive. These genes are important for lytic and persistent infection byviruses. These genes were isolated by generating gene trap libraries byinfecting cells with a retrovirus gene trap vector, selecting for cellsin which a gene trap event occurred (i.e., in which the vector hadinserted such that the promoterless marker gene was inserted such that acellular promoter promotes transcription of the marker gene, i.e.,inserted into a functioning gene), starving the cells of serum,infecting the selected cells with the virus of choice while continuingserum starvation, and adding back serum to allow visible colonies todevelop, which colonies were cloned by limiting dilution. Genes intowhich the retrovirus gene trap vector inserted were then isolated fromthe colonies using probes specific for the retrovirus gene trap vector.Thus nucleic acids isolated by this method are isolated portions ofgenes. Additionally, utilizing this method, several cellular genes wereisolated whose overexpression prevents viral infection or tumor growth,and they provide methods of treating viral infection or tumorgrowth/suppression by overexpression of these genes.

Thus the present invention provides a method of identifying a cellulargene necessary for viral growth in a cell and nonessential for cellularsurvival, comprising (a) transferring into a cell culture, e.g. growingin serum-containing medium, a vector encoding a selective marker genelacking a functional promoter, (b) selecting cells expressing the markergene, (c) removing serum from the culture medium, (d) infecting the cellculture with the virus, and (e) isolating from the surviving cells acellular gene within which the marker gene is inserted, therebyidentifying a gene necessary for viral growth in a cell and nonessentialfor cellular survival. The present invention also provides a method ofidentifying a cellular gene used for viral growth in a cell andnonessential for cellular survival, comprising (a) transferring into acell culture growing in serum-containing medium a vector encoding aselective marker gene lacking a functional promoter, (b) selecting cellsexpressing the marker gene, (c) removing serum from the culture medium,(d) infecting the cell culture with the virus, and (e) isolating fromthe surviving cells a cellular gene within which the marker gene isinserted, thereby identifying a gene necessary for viral growth in acell and nonessential for cellular survival or a gene whoseoverexpression prevents viral reproduction but is not fatal to thesurvival to the cell. In any selected cell type, such as Chinese hamsterovary cells, one can readily determine if serum starvation is requiredfor selection. If it is not, serum starvation may be eliminated from thesteps.

Alternatively, instead of removing serum from the culture medium, aserum factor required by the virus for growth can be inhibited, such asby the administration of an antibody that specifically binds thatfactor. Furthermore, if it is believed that there are no persistentlyinfected cells in the culture, the serum starvation step can beeliminated and the cells grown in usual medium for the cell type. Ifserum starvation is used, it can be continued for a time after theculture is infected with the virus. Serum can then be added back to theculture. If some other method is used to inactivate the factor, it canbe discontinued, inactivated or removed (such as removing theanti-factor antibody, e.g., with a bound antibody directed against thatantibody) prior to adding fresh serum back to the culture. Cells thatsurvive are mutants having an inactivating insertion in a gene necessaryfor growth of the virus. The genes having the insertions can then beisolated by isolating sequences having the marker gene sequences. Thismutational process disturbs a wild type function. A mutant gene mayproduce at a lower level a normal product, it may produce a normalproduct not normally found in these cells, it may cause theoverproduction of a normal product, it may produce an altered productthat has some functions but not others, or it may completely disrupt agene function. Additionally, the mutation may disrupt an RNA that has afunction but is never translated into a protein. For example, thealpha-tropomyosin gene has a 3′ RNA that is very important in cellregulation but never is translated into protein. (Cell 75 pg 1107-1117,Dec. 17, 1993).

As used herein, a cellular gene “nonessential for cellular survival”means a gene for which disruption of one or both alleles results in acell viable for at least a period of time which allows viral replicationto be inhibited for preventative or therapeutic uses or use in research.A gene “necessary for viral growth” means the gene product, eitherprotein or RNA, secreted or not, is necessary or beneficial, eitherdirectly or indirectly in some way for the virus to grow, and therefore,in the absence of that gene product (i.e., a functionally available geneproduct), the virus does not spread. For example, such genes can encodecell cycle regulatory proteins, proteins affecting the vacuolar hydrogenpump, or proteins involved in protein folding and protein modification,including but not limited to: phosphorylation, methylation,glycosylation, myristylation or other lipid moiety, or proteinprocessing via enzymatic processing. Some examples of such genes includevacuolar H+ATPase, alpha tropomyosin, gas5 gene, ras complex,N-acetyl-glucosaminy-l-transferase I MRNA, annexin II, c-golgi CM130 andcalcyclin.

Any virus capable of infecting the cell can be used for this method.Virus can be selected based upon the particular infection desired tostudy. However, it is contemplated by the present invention that manyviruses will be dependent upon the same cellular genes for survival;thus a cellular gene isolated using one virus can be used as a targetfor therapy for other viruses as well. Any cellular gene can be testedfor relevancy to any desired virus using the methods set forth herein,i.e., in general, by inhibiting the gene or its gene product in a celland determining if the desired virus can grow in that cell. Someexamples of viruses include HIV (including HIV-1 and HIV-2); parvovirus;papillomaviruses; hantaviruses; influenza viruses (e.g., influenza A, Band C viruses); hepatitis viruses A to G; caliciviruses; astroviruses;rotaviruses; coronaviruses, such as human respiratory coronavirus;picomaviruses, such as human rhinovirus and enterovirus; ebola virus;human herpesvirus (e.g., HSV-1-9); human adenovirus; for animal, theanimal counterpart to any above listed human virus, animal retroviruses,such as simian immunodeficiency virus, avian immunodeficiency virus,bovine immunodeficiency virus, feline immunodeficiency virus, equineinfectious anemia virus, caprine arthritis encephalitis virus,arenaviruses, arvoviruses, tickbone viruses or visna virus.

The nucleic acids comprising cellular genes of this invention wereisolated by the above method and as set forth in the examples. Theinvention includes a nucleic acid comprising the nucleotide sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:57,SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84,SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97,SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102,SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ IDNO:107, SEQ ID NO:108, SEQ ID NO:112,SEQ ID NO:120,SEQ ID NO:121,SEQ IDNO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, andSEQ ID NO:127 (this list is sometimes referred to herein as “SEQ LIST 1”for brevity). Thus these nucleic acids can contain, in addition to thenucleotides set forth in each SEQ ID NO in the sequence listing,additional nucleotides at either end of the molecule. Such additionalnucleotides can be added by any standard method, as known in the art,such as recombinant methods and synthesis methods. Examples of suchnucleic acids comprising the nucleotide sequence set forth in any entryof the sequence listing contemplated by this invention include, but arenot limited to, for example, the nucleic acid placed into a vector; anucleic acid having one or more regulatory region (e.g., promoter,enhancer, polyadenylation site) linked to it, particularly in functionalmanner, i.e. such that an mRNA or a protein can be produced; a nucleicacid including additional nucleic acids of the gene, such as a larger oreven full length genomic fragment of the gene, a partial or full lengthcDNA, a partial or full length RNA. Making and/or isolating such largernucleic acids is further described below and is well known and standardin the art.

Also provided in this invention are the double-stranded nucleic acidscorresponding to the nucleic acid sequences set forth in SEQ ID 1through SEQ ID 136, inclusive. It is recognized that “nucleic acid” asused herein, can refer to either or both strands of such double-strandednucleic acids, such strands often referred to as the “positive” and“negative” strands. Either strand of such double-stranded nucleic acidsmay encode the polypeptides of this invention, and the coding sequencesfor such polypeptides encoded by either strand are disclosed herein.

The invention also provides a nucleic acid encoding the protein encodedby the gene comprising the nucleotide sequence set forth in any of thesequences listed in SEQ LIST 1, as well as allelic variants and homologsof each such gene. The gene is readily obtained using standard methods,as described below and as is known and standard in the art. The presentinvention also contemplates any unique fragment of these genes or of thenucleic acids set forth in any of the sequences listed in SEQ LIST 1.Examples of inventive fragments of the inventive genes can include thenucleic acids whose sequence is set forth in any of the sequences listedin SEQ LIST 1. To be unique, the fragment must be of sufficient size todistinguish it from other known sequences, most readily determined bycomparing any nucleic acid fragment to the nucleotide sequences ofnucleic acids in computer databases, such as GenBank. Such comparativesearches are standard in the art. Typically, a unique fragment useful asa primer or probe will be at least about 20 to about 25 nucleotides inlength, depending upon the specific nucleotide content of the sequence.Additionally, fragments can be, for example, at least about 30, 40, 50,75, 100, 200 or 500 nucleotides in length. The nucleic acids can besingle or double stranded, depending upon the purpose for which it isintended.

The present invention further provides a nucleic acid comprising theregulatory region of a gene comprising any one of the nucleotidesequences set forth in SEQ LIST 1, as well as homologs of each suchgene. Additionally provided is a construct comprising such a regulatoryregion functionally linked to a reporter gene. Such reporter geneconstructs can be used to screen for compounds and compositions thataffect expression of the gene comprising the nucleic acids whosesequence is set forth in SEQ LIST 1, or any homologs thereof.

The nucleic acids set forth in the sequence listing are gene fragments;the entire coding sequence and the entire gene that comprises eachfragment are both contemplated herein and are readily obtained bystandard methods, given the nucleotide sequences presented in thesequence listing (see. e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989; DNA cloning: A Practical Approach, Volumes I and II,Glover, D. M. ed., IRL Press Limited, Oxford, 1985). To obtain theentire genomic gene, briefly, a nucleic acid whose sequence is set forthin any of SEQ ID NO: 1 through SEQ ID NO: 127, or preferably in any ofthe sequences listed in SEQ LIST 1, or a smaller fragment thereof, isutilized as a probe to screen a genomic library under high stringencyconditions, and isolated clones are sequenced. Once the sequence of thenew clone is determined, a probe can be devised from a portion of thenew clone not present in the previous fragment and hybridized to thelibrary to isolate more clones containing fragments of the gene. In thismanner, by repeating this process in organized fashion, one can “walk”along the chromosome and eventually obtain nucleotide sequence for theentire gene. Similarly, one can use portions of the present fragments,or additional fragments obtained from the genomic library, that containopen reading frames to screen a cDNA library to obtain a cDNA having theentire coding sequence of the gene. Repeated screens can be utilized asdescribed above to obtain the complete sequence from several clones ifnecessary. The isolates can then be sequenced to determine thenucleotide sequence by standard means such as dideoxynucleotidesequencing methods (see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989).

The present genes were isolated from rat; however, homologs in anydesired species, preferably mammalian, such as human, can readily beobtained by screening a human library, genomic or cDNA, with a probecomprising sequences of the nucleic acids set forth in the sequencelisting herein, or fragments thereof, and isolating genes specificallyhybridizing with the probe under preferably relatively high stringencyhybridization conditions. For example, high salt conditions (e.g., in6×SSC or 6×SSPE) and/or high temperatures of hybridization can be used.For example, the stringency of hybridization is typically about 5° C. to20° C. below the T_(m) (the melting temperature at which half of themolecules dissociate from its partner) for the given chain length. As isknown in the art, the nucleotide composition of the hybridizing regionfactors in determining the melting temperature of the hybrid. For 20merprobes, for example, the recommended hybridization temperature istypically about 55-58° C. Additionally, the rat sequence can be utilizedto devise a probe for a homolog in any specific animal by determiningthe amino acid sequence for a portion of the rat protein, and selectinga probe with optimized codon usage to encode the amino acid sequence ofthe homolog in that particular animal. Any isolated gene can beconfirmed as the targeted gene by sequencing the gene to determine itcontains the nucleotide sequence listed herein as comprising the gene.Any homolog can be confirmed as a homolog by its functionality.

Additionally contemplated by the present invention are nucleic acids,from any desired species, preferably mammalian and more preferablyhuman, having 98%, 95%, 90%, 85%, 80%, 70%, 60%, or 50% homology, orgreater, in the region of homology, to a region in an exon of a nucleicacid encoding the protein encoded by the gene comprising the nucleotidesequence set forth in any of the sequences listed in SEQ LIST 1 or tohomologs thereof. Also contemplated by the present invention are nucleicacids, from any desired species, preferably mammalian and morepreferably human, having 98%, 95%, 90%, 85%, 80%, 70%, 60%, or 50%homology, or greater, in the region of homology, to a region in an exonof a nucleic acid comprising the nucleotide sequence set forth in any ofthe sequences listed in SEQ LIST 1 or to homologs thereof. These genescan be synthesized or obtained by the same methods used to isolatehomologs, with stringency of hybridization and washing, if desired,reduced accordingly as homology desired is decreased, and further,depending upon the G-C or A-T richness of any area wherein variabilityis searched for. Allelic variants of any of the present genes or oftheir homologs can readily be isolated and sequenced by screeningadditional libraries following the protocol above. Methods of makingsynthetic genes are described in U.S. Pat. No. 5,503,995 and thereferences cited therein.

The nucleic acid encoding any selected protein of the present inventioncan be any nucleic acid that functionally encodes that protein. Forexample, to functionally encode, i.e., allow the nucleic acid to beexpressed, the nucleic acid can include, for example, exogenous orendogenous expression control sequences, such as an origin ofreplication, a promoter, an enhancer, and necessary informationprocessing sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences can be promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. Expression control sequencescan be selected for functionality in the cells in which the nucleic acidwill be placed. A nucleic acid encoding a selected protein can readilybe determined based upon the amino acid sequence of the selectedprotein, and, clearly, many nucleic acids will encode any selectedprotein.

The present invention additionally provides a nucleic acid thatselectively hybridizes under stringent conditions with a nucleic acidset forth in SEQ LIST 1 or with a nucleic acid encoding the proteinencoded by the gene comprising the nucleotide sequence set forth in anysequence listed in SEQ LIST 1. This hybridization can be specific. Thedegree of complementarity between the hybridizing nucleic acid and thesequence to which it hybridizes should be at least enough to excludehybridization with a nucleic acid encoding an unrelated protein. Thus, anucleic acid that selectively hybridizes with a nucleic acid of thepresent protein coding sequence will not selectively hybridize understringent conditions with a nucleic acid for a different, unrelatedprotein, and vice versa. Typically, the stringency of hybridization toachieve selective hybridization involves hybridization in high ionicstrength solution (6×SSC or 6×SSPE) at a temperature that is about12-25° C. below the T_(m) (the melting temperature at which half of themolecules dissociate from its partner) followed by washing at acombination of temperature and salt concentration chosen so that thewashing temperature is about 5° C. to 20° C. below the T_(m) of thehybrid molecule. The temperature and salt conditions are readilydetermined empirically in preliminary experiments in which samples ofreference DNA immobilized on filters are hybridized to a labeled nucleicacid of interest and then washed under conditions of differentstringencies. Hybridization temperatures are typically higher forDNA-RNA and RNA-RNA hybridizations. The washing temperatures can be usedas described above to achieve selective stringency, as is known in theart. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Kunkel etal. Methods Enzymol. 1987:154:367, 1987). Nucleic acid fragments thatselectively hybridize to any given nucleic acid can be used, e.g., asprimers and or probes for further hybridization or for amplificationmethods (e.g., polymerase chain reaction (PCR), ligase chain reaction(LCR)) . A preferable stringent hybridization condition for a DNA:DNAhybridization can be at about 68° C. (in aqueous solution) in 6×SSC or6×SSPE followed by washing at 68° C.

The present invention additionally provides a polypeptide comprising theamino acid sequence encoded by the gene comprising the nucleotidesequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:56,SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:77,SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83,SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88,SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96,SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ IDNO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO: 120, SEQID NO:121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO: 125,SEQ ID NO:126, and SEQ ID NO: 127 (i.e . . , SEQ LIST 1). Additionally,polypeptides comprising the amino acid sequence encoded by a nucleicacid that selectively hybridizes under stringent conditions with anucleic acid in SEQ LIST 1 are provided. Further, polypeptidescomprising the amino acid sequence encoded by a nucleic acid having aregion within an exon wherein the region has at least 50, 60, 70, 80,90, or 95% homology with a nucleic acid in SEQ LIST 1. Thesepolypeptides can be readily obtained by any of several means. Forexample, the nucleotide sequence of coding regions of the gene can betranslated and then the corresponding polypeptide can be synthesizedmechanically by standard methods. Additionally, the coding regions ofthe genes can be expressed or synthesized, an antibody specific for theresulting polypeptide can be raised by standard methods (see, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988), and the protein can beisolated from other cellular proteins by selective hybridization withthe antibody. This protein can be purified to the extent desired bystandard methods of protein purification (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). The amino acid sequence ofany protein, polypeptide or peptide of this invention can be deducedfrom the nucleic acid sequence, or it can be determined by sequencing anisolated or recombinantly produced protein.

The terms “peptide,” “polypeptide” and “protein” can be usedinterchangeably herein and refer to a polymer of amino acids andincludes full-length proteins and fragments thereof. As used in thespecification and in the claims, “a” can mean one or more, dependingupon the context in which it is used. An amino acid residue is an aminoacid formed upon chemical digestion (hydrolysis) of a polypeptide at itspeptide linkages. The amino acid residues described herein arepreferably in the L isomeric form. However, residues in the D isomericform can be substituted for any L-amino acid residue, as long as thedesired functional property is retained by the polypeptide. Standardpolypeptide nomenclature (described in J. Biol. Chem., 243:3552-59(1969) and adopted at 37 CFR § 1.822(b)) is used herein.

As will be appreciated by those skilled in the art, the invention alsoincludes those polypeptides having slight variations in amino acidsequences or other properties. Amino acid substitutions can be selectedby known parameters to be neutral (see, e.g., Robinson W E Jr, andMitchell W M., AIDS 4:S151-S162(1990)). Such variations may arisenaturally as allelic variations (e.g., due to genetic polymorphism) ormay be produced by human intervention (e.g., by mutagenesis of clonedDNA sequences), such as induced point, deletion, insertion andsubstitution mutants. Minor changes in amino acid sequence are generallypreferred, such as conservative amino acid replacements, small internaldeletions or insertions, and additions or deletions at the ends of themolecules. Substitutions may be designed based on, for example, themodel of Dayhoff, et al. (in Atlas of Protein Sequence and Structure1978, Nat'l Biomed. Res. Found., Washington, D.C.). These modificationscan result in changes in the amino acid sequence, provide silentmutations, modify a restriction site, or provide other specificmutations. Likewise, such amino acid changes result in a differentnucleic acid encoding the polypeptides and proteins. Thus, alternativenucleic acids are also contemplated by such modifications.

The present invention also provides cells containing a nucleic acid ofthe invention. A cell containing a nucleic acid encoding a proteintypically can replicate the DNA and, further, typically can express theencoded protein. The cell can be a prokaryotic cell, particularly forthe purpose of producing quantities of the nucleic acid, or a eukaryoticcell, particularly a mammalian cell. The cell is preferably a mammaliancell for the purpose of expressing the encoded protein so that theresultant produced protein has mammalian protein processingmodifications.

Nucleic acids of the present invention can be delivered into cells byany selected means, in particular depending upon the purpose of thedelivery of the compound and the target cells. Many delivery means arewell-known in the art. For example, electroporation, calcium phosphateprecipitation, microinjection, cationic or anionic liposomes, andliposomes in combination with a nuclear localization signal peptide fordelivery to the nucleus can be utilized, as is known in the art.

The present invention also contemplates that the mutated cellular genesnecessary for viral growth, produced by the present method, as well ascells containing these mutants can also be useful. These mutated genesand cells containing them can be isolated and/or produced according tothe methods herein described and using standard methods.

It should be recognized that the sequences set forth herein may containminor sequencing errors. Such errors can be corrected, for example, byusing the hybridization procedure described above with various probesderived from the described sequences such that the coding sequence canbe reisolated and resequenced.

As described in the examples, the present invention provides thediscovery of a “serum survival factor” present in serum that isnecessary for the survival of persistently virally infected cells.Isolation and characterization of this factor have shown it to be aprotein, to have a molecular weight of between about 50 kD and 100 kD,to resist inactivation in low pH (e.g., pH2) and chloroform extraction,to be inactivated by boiling for about 5 minutes and in low ionicstrength solution (e.g., about 10 mM to about 50 mM). The presentinvention thus provides a purified mammalian serum protein having amolecular weight of between about 50 kD and 100 kD which resistsinactivation in low pH and resists inactivation by chloroformextraction, which inactivates when boiled and inactivates in low ionicstrength solution, and which when removed from a cell culture comprisingcells persistently infected with reovirus selectively substantiallyprevents survival of cells persistently infected with reovirus. Thefactor, fitting the physical characteristics described above, canreadily be verified by adding it to non-serum-containing medium (whichpreviously could not support survival of persistently virally infectedcells) and determining whether this medium with the added putativefactor can now support persistently virally infected cells, particularlycells persistently infected with reovirus. As used herein, a “purified”protein means the protein is at least of sufficient purity such that anapproximate molecular weight can be determined.

The amino acid sequence of the protein can be elucidated by standardmethods. For example, an antibody to the protein can be raised and usedto screen an expression library to obtain nucleic acid sequence codingthe protein. This nucleic acid sequence is then simply translated intothe corresponding amino acid sequence. Alternatively, a portion of theprotein can be directly sequenced by standard amino acid sequencingmethods (amino-terminus sequencing). This amino acid sequence can thenbe used to generate an array of nucleic acid probes that encompasses allpossible coding sequences for a portion of the amino acid sequence. Thearray of probes is used to screen a cDNA library to obtain the remainderof the coding sequence and thus ultimately the corresponding amino acidsequence.

The present invention also provides methods of detecting and isolatingadditional serum survival factors. For example, to determine if anyknown serum components are necessary for viral growth, the knowncomponents can be inhibited in, or eliminated from, the culture medium,and it can be observed whether viral growth is inhibited by determiningif persistently infected cells do not survive. One can add the factorback (or remove the inhibition) and determine whether the factor allowsfor viral growth.

Additionally, other, unknown serum components can also be found to beessential for growth. Serum can be fractionated by various standardmeans, and fractions added to serum free medium to determine if a factoris present in a reaction that allows growth previously inhibited by thelack of serum. Fractions having this activity can then be furtherfractionated until the factor is relatively free of other components.The factor can then be characterized by standard methods, such as sizefractionation, denaturation and/or inactivation by various means, etc.Preferably, once the factor has been purified to a desired level ofpurity, it is added to cells in serum free medium to confirm that itbestows the function of allowing virus to grow when serum-free mediumalone did not. This method can be repeated to confirm the requirementfor the specific factor for any desired virus, since each serum factorfound to be required by any one virus can also be required by many otherviruses. In general, the closer the viruses are related and the moresimilar the infection modes of the viruses, the more likely that afactor required by one virus will be required by the other.

The present invention also provides methods of treating virus infectionsutilizing applicants' discoveries. The subject of any of the hereindescribed methods can be any animal, preferably a mammal, such as ahuman, a veterinary animal, such as a cat, dog, horse, pig, goat, sheep,or cow, or a laboratory animal, such as a mouse, rat, rabbit, or guineapig, depending upon the virus.

The present invention provides a method of reducing or inhibiting, andthereby treating, a viral infection in a subject, comprisingadministering to the subject an inhibiting amount of a composition thatinhibits functioning of the serum protein described herein, i.e. theserum protein having a molecular weight of between about 50 kD and 100kD which resists inactivation in low pH and resists inactivation bychloroform extraction, which inactivates when boiled and inactivates inlow ionic strength solution, and which when removed from a cell culturecomprising cells persistently infected with the virus prevents survivalof at least some cells persistently infected with the virus, therebytreating the viral infection. The composition can comprise, for example,an antibody that specifically binds the serum protein, or an antisenseRNA that binds an RNA encoded by a gene functionally encoding the serumprotein.

Any virus capable of infecting the selected subject to be treated can betreated by the present methods. As described above, any serum protein orsurvival factor found by the present methods to be necessary for growthof cells infected with any one virus can be found to be necessary forgrowth of the cells infected with many other viruses. For any givencell-virus combination, the serum protein or factor can be confirmed tobe required for growth by the methods described herein. The cellulargenes identified by the examples using reovirus, a mammalian pathogen,and a rat cell system have general applicability to other virusinfections that include all of the known as well as yet to be discoveredhuman pathogens, including, but not limited to: human immunodeficiencyviruses (e.g., HIV-1, HIV-2); parvovirus; papillomaviruses;hantaviruses; influenza viruses (e.g., influenza A, B and C viruses);hepatitis viruses A to G; caliciviruses; astroviruses; rotaviruses;coronaviruses, such as human respiratory coronavirus; picomaviruses,such as human rhinovirus and enterovirus; ebola virus; human herpesvirus(e.g., HSV-1-9); human adenovirus; hantaviruses; for animal, the animalcounterpart to any above listed human virus, animal retroviruses, suchas simian immunodeficiency virus, avian immunodeficiency virus, bovineimmunodeficiency virus, feline immunodeficiency virus, equine infectiousanemia virus, caprine arthritis encephalitis virus, arenaviruses,arvoviruses, tickbone virus or visna virus.

A protein inhibiting amount of the composition can be readilydetermined, such as by administering varying amounts to cells or to asubject and then adjusting the effective amount for inhibiting theprotein according to the volume of blood or weight of the subject.Compositions that bind to the protein can be readily determined byrunning the putatively bound protein on a protein gel and observing analteration in the protein's migration through the gel. Inhibition of theprotein can be determined by any desired means such as adding theinhibitor to complete media used to maintain persistently infected cellsand observing the cells' viability. The composition can comprise, forexample, an antibody that specifically binds the serum protein. Specificbinding by an antibody means that the antibody can be used toselectively remove the factor from serum or inhibit the factor'sbiological activity and can readily be determined by radio immune assay(RIA), bioassay, or enzyme-linked immunosorbant (ELISA) technology. Thecomposition can comprise, for example, an antisense RNA thatspecifically binds an RNA encoded by the gene encoding the serumprotein. Antisense RNAs can be synthesized and used by standard methods(e.g., Antisense RNA and DNA, D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1988)).

The present methods provide a method of screening a compound foreffectiveness in treating or preventing a viral infection, comprisingadministering the compound to a cell containing a cellular genefunctionally encoding a gene product necessary for reproduction of thevirus in the cell but not necessary for survival of the cell anddetecting the level and/or activity (i.e. function) of the gene productproduced, a decrease or elimination of the gene product and/or the geneproduct activity indicating a compound for treating or preventing theviral infection. The cellular gene can be, for example, a nucleic acidset forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57,SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77,SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82;SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87,SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO: 111, SEQ ID NO: 112,SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ IDNO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO:121,SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ IDNO:126, or SEQ ID NO:127 (herein sometimes referred to as SEQ LIST 2,for brevity), any homolog thereof, or any other gene obtained using themethods provided herein for obtaining such genes. It is understood thatthe cellular gene can be present naturally in the cell being screened,or it can be introduced into the cell in a suitable expression vector,as are well known in the art. The level of the gene product can bemeasured by any standard means, such as by detection with an antibodyspecific for the protein. The level of gene product can be compared tothe level of the gene product in a control cell not contacted with thecompound. The level of gene product can be compared to the level of thegene product in the same cell prior to addition of the compound.Activity, or function, can be measured by any standard means, such as byenzymatic assays that measure the conversion of a substrate to a productor binding assays that measure the binding of a protein to a nucleicacid, for example. Examples of gene products disclosed herein whoseactivity/function can be measured include tristetraprolin (humanZFP-36), 6-pyruvoyl-tetrahydropterin synthase, a eukaryotic DnaJ-likeprotein, ID3 (inhibitor of DNA binding 3),N-acetylglucos-aminyltransferase I (mGAT-1), cleavage stimulation factor(CSTF2), TAK1 binding protein, human zinc transcription factor ZPF207,Dlx2, Smad7 (Mad-related protein), and P-glycoprotein (mdrlb). Theactivity can be compared to the activity in a control cell not contactedwith the compound or in the same cell prior to addition of the compound.Relatedly, the regulatory region of the gene can be functionally linkedto a reporter gene and compounds can be screened for inhibition of thereporter gene. Such reporter constructs are described herein.

The present invention also provides a method of screening a compound foreffectiveness in treating or preventing a viral infection comprisingcontacting the compound with the gene product of a cellular genecomprising a nucleic acid of SEQ LIST 2, or any homolog thereof, anddetecting the function of the gene product, a decrease or elimination ofthe function indicating a compound effective for treating or preventingviral infection. Examples of gene products disclosed herein that can beutilized in this method include tristetraprolin (human ZFP-36),6-pyruvoyl-tetrahydropterin synthase, a eukaryotic DnaJ-like protein,ID3 (inhibitor of DNA binding 3), N-acetylglucos-aminyltransferase I(mGAT-1), cleavage stimulation factor (CSTF2), TAKI binding protein,human zinc transcription factor ZPF207, Dlx2, Smad7 (Mad-relatedprotein), and P-glycoprotein (mdrlb).

The present invention provides a method of selectively eliminating cellspersistently infected with a virus from an animal cell culture capableof surviving for a first period of time in the absence of serum,comprising propagating the cell culture in the absence of serum for asecond time period during which a persistently infected cell cannotsurvive without serum, thereby selectively eliminating from the cellculture cells persistently infected with the virus. The second timeperiod should be shorter than the first time period. Thus one can simplyeliminate serum from a standard culture medium composition for a periodof time (e.g. by removing serum containing medium from the culturecontainer, rinsing the cells, and adding serum-free medium back to thecontainer), then, after a time of serum starvation, return serum to theculture medium. Alternatively, one can inhibit a serum survival factorfrom the culture in place of the step of serum starvation. Furthermore,one can instead interfere with the virus-factor interaction. Such aviral elimination method can periodically be performed for culturedcells to ensure that they remain virus-free. The time period of serumremoval can greatly vary, with a typical range being about 1 to about 30days; a preferable period can be about 3 to about 10 days, and a morepreferable period can be about 5 days to about 7 days. This time periodcan be selected based upon ability of a specific cell to survive withoutserum as well as the life cycle of the target virus, e.g., forreovirus,. which has a life cycle of about 24 hours, 3 days' starvationof cells provides dramatic results.

Furthermore, the time period can be shortened by also passaging thecells during the starvation; in general, increasing the number ofpassages can decrease the time of serum starvation (or serum factorinhibition) needed to get full clearance of the virus from the culture.While passaging, the cells typically are exposed briefly to serum(typically for about 3 to about 24 hours). This exposure both stops theaction of the trypsin used to dislodge the cells and stimulates thecells into another cycle of growth, thus aiding in this selectionprocess. Thus a starvation/serum cycle can be repeated to optimize theselective effect. Other standard culture parameters, such as confluencyof the cultures, pH, temperature, etc. can be varied to alter the neededtime period of serum starvation (or serum survival factor inhibition).This time period can readily be determined for any given viral infectionby simply removing the serum for various periods of time, then testingthe cultures for the presence of the infected cells (e.g., by ability tosurvive in the absence of serum and confirmed by quantitating virus incells by standard virus titration and immunohistochemical techniques) ateach tested time period, and then detecting at which time periods ofserum deprivation the virally infected cells were eliminated. It ispreferable that shorter time periods of serum deprivation that stillprovide elimination of the persistently infected cells be used.Furthermore, the cycle of starvation, then adding back serum anddetermining amount of virus remaining in the culture can be repeateduntil no virtually infected cells remain in the culture.

Thus, the present method can further comprise passaging the cells, i.e.,transferring the cell culture from a first container to a secondcontainer. Such transfer can facilitate the selective lack of survivalof virally infected cells. Transfer can be repeated several times.Transfer is achieved by standard methods of tissue culture (see, e.g.,Freshney, Culture of Animal Cells, A Manual of Basic Technique,2nd Ed.Alan R. Liss, Inc., New York, 1987).

The present method further provides a method of selectively eliminatingfrom a cell culture cells persistently infected with a virus, comprisingpropagating the cell culture in the absence of a functional form of theserum protein having a molecular weight of between about 50 kD and 100kD which resists inactivation in low pH and resists inactivation bychloroform extraction, which inactivates when boiled and inactivates inlow ionic strength solution, and which when removed from a cell culturecomprising cells persistently infected with reovirus substantiallyprevents survival of cells persistently infected with reovirus. Theabsence of the functional form can be achieved by any of severalstandard means, such as by binding the protein to an antibody selectivefor it (binding the antibody in serum either before or after the serumis added to the cells; if before, the serum protein can be removed fromthe serum by, e.g., binding the antibody to a column and passing theserum over the column and then administering the survival protein-freeserum to the cells), by administering a compound that inactivates theprotein, or by administering a compound that interferes with theinteraction between the virus and the protein.

Thus, the present invention provides a method of selectively eliminatingfrom a cell culture propagated in serum-containing medium cellspersistently infected with a virus, comprising inhibiting in the serumthe protein having a molecular weight of between about 50 kD and 100 kDwhich resists inactivation in low pH and resists inactivation bychloroform extraction, which inactivates when boiled and inactivates inlow ionic strength solution, and which when removed from a cell culturecomprising cells persistently infected with reovirus substantiallyprevents survival of cells persistently infected with reovirus.Alternatively, the interaction between the virus and the serum proteincan be disrupted to selectively eliminate cells persistently infectedwith the virus.

Any virus capable of some form of persistent infection may be eliminatedfrom a cell culture utilizing the present elimination methods, includingremoving, inhibiting or otherwise interfering with a serum protein, suchas the one exemplified herein, and also including removing, inhibitingor otherwise interfering with a gene product from any cellular genefound by the present method to be necessary for viral growth yetnonessential to the cell. For example, DNA viruses or RNA viruses can betargeted. One can readily determine whether cells infected with aselected virus can be selectively removed from a culture through removalof serum by starving cells permissive to the virus of serum (orinhibiting the serum survival factor), adding the selected virus to thecells, adding serum to the culture, and observing whether infected cellsdie (i.e., by titering levels of virus in the surviving cells with anantibody specific for the virus).

A culture of any animal cell (i.e., any cell that is typically grown andmaintained in culture in serum) that can be maintained for a period oftime in the absence of serum, can be purified from viral infectionutilizing the present method. For example, primary cultures as well asestablished cultures and cell lines can be used. Furthermore, culturesof cells from any animal and any tissue or cell type within that animalthat can be cultured and that can be maintained for a period of time inthe absence of serum can be used. For example, cultures of cells fromtissues typically infected, and particularly persistently infected, byan infectious virus could be used.

As used in the claims “in the absence of serum” means at a level atwhich persistently virally infected cells do not survive. Typically, thethreshold level is about 1% serum in the media. Therefore, about 1%serum or less can be used, such as about 1%, 0.75%, 0.50% . 0.25% 0.1%or no serum can be used.

As used herein, “selectively eliminating” cells persistently infectedwith a virus means that substantially all of the cells persistentlyinfected with the virus are killed such that the presence of virallyinfected cells cannot be detected in the culture immediately after theelimination procedure has been performed. Furthermore, “selectivelyeliminating” includes that cells not infected with the virus aregenerally not killed by the method. Some surviving cells may stillproduce virus but at a lower level, and some may be defective inpathways that lead to death by the virus. Typically, for cellspersistently infected with virus to be substantially all killed, morethan about 90% of the cells, and more preferably more than about 95%,98%, 99%, or 99.99% of virus-containing cells in the culture are killed.

The present method also provides a nucleic acid comprising theregulatory region of any of the genes. Such regulatory regions can beisolated from the genomic sequences isolated and sequenced as describedabove and identified by any characteristics observed that arecharacteristic for regulatory regions of the species and by theirrelation to the start codon for the coding region of the gene. Thepresent invention also provides a construct comprising the regulatoryregion functionally linked to a reporter gene. Such constructs are madeby routine subcloning methods, and many vectors are available into whichregulatory regions can be subcloned upstream of a marker gene. Markergenes can be chosen for ease of detection of marker gene product.

The present method therefore also provides a method of screening acompound for treating a viral infection, comprising administering thecompound to a cell containing any of the above-described constructs,comprising a regulatory region of one of the genes comprising any of thenucleotide sequences set forth in SEQ LIST 2, or any homologs thereof,whose inhibition or reduction in expression causes inhibition of viralreplication wherein the region is functionally linked to a reportergene, and detecting the level of the reporter gene product produced, adecrease or elimination of the reporter gene product indicating acompound for treating the viral infection. Compounds detected by thismethod would inhibit transcription of the gene from which the regulatoryregion was isolated, and thus, in treating a subject, would inhibit theproduction of the gene product produced by the gene, and thus treat theviral infection.

Some genes when disrupted by the present method of retrovirus insertion,resulted in over expression of the gene product, and this overexpressioninhibited viral replication. Thus the present invention provides amethod of screening a compound for effectiveness in treating a viralinfection, comprising administering the compound to a cell containing acellular gene functionally encoding a gene product whose overexpressioninhibits reproduction of the virus but does not prevent survival of thecell and detecting the level of the gene product produced, an increasein the gene product indicating a compound effective for treating theviral infection. Typically, an increase will be a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%or higher increase over gene product produced when the compound is notpresent.

The present invention additionally provides a method of reducing orinhibiting a viral infection in a subject, comprising administering tothe subject an amount of a composition that inhibits expression orfunctioning of a gene product encoded by a gene comprising the nucleicacid set forth in any of SEQ LIST 2, or a homolog thereof, therebytreating the viral infection. Reducing or inhibiting viral infectionnaturally can include both the initial infection of the subject and theinfection of uninfected cells within an already infected subject, e.g.inhibiting viral replication in cells of the subject. The compositioncan comprise, for example, an antibody that binds a protein encoded bythe gene. The composition can also comprise an antibody that binds areceptor for a protein encoded by the gene. Such an antibody can beraised against the selected protein by standard methods as set forthabove, and can be either polyclonal or monoclonal, though monoclonal ispreferred. Alternatively, the composition can comprise an antisense RNAthat binds an RNA encoded by the gene, as described above. Examples ofantisense RNA useful therapeutically include the fragments of thenucleic acids described above. Furthermore, the composition can comprisea nucleic acid functionally encoding an antisense RNA that binds an RNAencoded by the gene. Other useful compositions will be readily apparentto the skilled artisan.

The present invention also provides a method of treating a viralinfection in a subject comprising administering to the subject atreatment effective amount of a composition that increases expression ofa gene whose over expression reduces or inhibits viral replication.Typically, an increase will be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or higher increaseover gene product produced when the composition is not present.

The present invention further provides a method of reducing orinhibiting a viral infection in a subject comprising mutating ex vivo ina selected cell, for example from the subject or from an allogenicsource, an endogenous gene comprising a nucleic acid set forth in SEQLIST 2 whose inhibition or reduction in expression causes inhibition ofviral replication, or a homolog thereof, to a gene form incapable ofproducing a functional gene product of the gene or a gene form producinga reduced amount of a functional gene product of the gene, and placing(or replacing, in the case of the subject's own cells) the cell in thesubject, thereby reducing viral infection of cells in the subject. Thecell can be selected according to the typical target cell of thespecific virus whose infection is to be reduced, prevented or inhibited.A preferred cell for several viruses is a hematopoietic cell. When theselected cell is a hematopoietic cell, viruses which can be reduced orinhibited from infection can include, for example, HIV, including HIV- 1and HIV-2. However, many other virus-cell combinations will be apparentto the skilled artisan.

The invention also includes a method of reducing or inhibiting viralinfection in a subject comprising mutating ex vivo in a selected cell,for example from a subject or an allogenic source, an endogenous genecomprising a nucleic acid set forth in SEQ LIST 2 whose overexpressioncauses inhibition of viral replication, or a homolog thereof, to a geneform that expresses the gene at a higher level than the endogenous gene,and placing or replacing the cell in the subject. Typically, a higherlevel can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%,150%, 175%, 200%, 300%, 400%, 500% or higher than the non-mutated,endogenous gene. The cell can be selected according to the typicaltarget cell of the specific virus whose infection is to be reduced,prevented or inhibited. A preferred cell for several viruses is ahematopoietic cell. When the selected cell is a hematopoietic cell,viruses which can be reduced or inhibited from infection can include,for example, HIV, including HIV-1 and HIV-2. However, many othervirus-cell combinations will be apparent to the skilled artisan.

The present invention additionally provides a method of increasing viralinfection resistance in a subject comprising mutating ex vivo in aselected cell, for example from the subject or from an allogenic source,an endogenous gene comprising a nucleic acid set forth in SEQ LIST 2,whose inhibition or reduction in expression increases viral infectionresistance, said endogenous gene being mutated to a mutated gene formincapable of producing a functional gene product of the gene or a geneform producing a reduced amount of a functional gene product of thegene, and placing the cell in the subject, thereby increasing viralinfection resistance of cells in the subject. The virus can be HIV,particularly when the cell is a hematopoietic cell. However, many othervirus-cell combinations will be apparent to the skilled artisan.

Furthermore, the present invention provides a method for isolation ofcellular genes utilized in tumor progression. The present inventionprovides a method of identifying a cellular gene that can suppress amalignant phenotype in a cell, comprising (a) transferring into a cellculture incapable of growing well in soft agar or Matrigel a vectorencoding a selective marker gene lacking a functional promoter, (b)selecting cells expressing the marker gene, and (c) isolating fromselected cells which are capable of growing in soft agar or Matrigel acellular gene within which the marker gene is inserted, therebyidentifying a gene that can suppress a malignant phenotype in a cell.This method can be performed using any selected non-transformed cellline, of which many are known in the art.

The present invention additionally provides a method of identifying acellular gene that can suppress a malignant phenotype in a cell,comprising (a) transferring into a cell culture of non-transformed cellsa vector encoding a selective marker gene lacking a functional promoter,(b) selecting cells expressing the marker gene, and (c) isolating fromselected and transformed cells a cellular gene within which the markergene is inserted, thereby identifying a gene that can suppress amalignant phenotype in a cell. A non-transformed phenotype can bedetermined by any of several standard methods in the art, such as theexemplified inability to grow in soft agar, or inability to grow inMatrigel.

The present invention further provides a method of screening for acompound for suppressing a malignant phenotype in a cell comprisingadministering the compound to a cell containing a cellular genefunctionally encoding a gene product involved in establishment of amalignant phenotype in the cell and detecting the level of the geneproduct produced, a decrease, inhibition or elimination of the geneproduct indicating a compound effective for suppressing the malignantphenotype. Detection of the level, or amount, of gene product producedcan be measured, directly or indirectly, by any of several methodsstandard in the art (e.g., protein gel, antibody-based assay, detectinglabeled RNA) for assaying protein levels or amounts, and selected basedupon the specific gene product.

The present invention also provides a method of screening for a compoundfor suppressing a malignant phenotype in a cell comprising administeringthe compound to a cell containing a cellular gene functionally encodinga gene product whose overexpression is involved in suppressing amalignant phenotype in the cell and detecting the level of the geneproduct produced, an increase in the gene product indicating a compoundeffective for suppressing the malignant phenotype.

The present invention further provides a method of suppressing amalignant phenotype in a cell in a subject, comprising administering tothe subject an amount of a composition that inhibits expression orfunctioning of a gene product encoded by a gene comprising the nucleicacid set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:36 or SEQ ID NO:94, or a homologthereof, or any gene whose overexpression is found by the present methodto be involved in suppressing a malignant phenotype in the cell (e.g.,any clone designated herein with an “x”) thereby suppressing a malignantphenotype. The composition can, for example, comprise an antibody thatbinds a protein encoded by the gene. The composition can, as anotherexample, comprise an antibody that binds a receptor for a proteinencoded by the gene. The composition can comprise an antisense RNA thatbinds an RNA encoded by the gene. Further, the composition can comprisea nucleic acid functionally encoding an antisense RNA that binds an RNAencoded by the gene.

The present invention further provides a method of suppressing amalignant phenotype in a cell in a subject, comprising administering tothe subject an amount of a composition that increases expression of agene product whose overexpression is involved in suppressing a malignantphenotype in the cell. The gene product can be the product of a genewherein disruption of an upstream gene by the present vector resulted inoverexpression of the downstream gene, and the overexpression of thedownstream gene demonstrated a transformed phenotype. The compositioncan be, for example, an inhibitor, such as a small molecule inhibitor,of the COX 2 enzyme.

Diagnostic or therapeutic agents of the present invention can beadministered to a subject or an animal model by any of many standardmeans for administering therapeutics or diagnostics to that selectedsite or standard for administering that type of functional entity. Forexample, an agent can be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, topically, transdermally, or the like. Agents can beadministered, e.g., as a complex with cationic liposomes, orencapsulated in anionic liposomes. Compositions can include variousamounts of the selected agent in combination with a pharmaceuticallyacceptable carrier and, in addition, if desired, may include othermedicinal agents, pharmaceutical agents, carriers, adjuvants, diluents,etc. Parental administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Depending uponthe mode of administration, the agent can be optimized to avoiddegradation in the subject, such as by encapsulation, etc.

Dosages will depend upon the mode of administration, the disease orcondition to be treated, and the individual subject's condition, butwill be that dosage typical for and used in administration of antiviralor anticancer agents. Dosages will also depend upon the compositionbeing administered, e.g., a protein or a nucleic acid. Such dosages areknown in the art. Furthermore, the dosage can be adjusted according tothe typical dosage for the specific disease or condition to be treated.Furthermore, viral titers in culture cells of the target cell type canbe used to optimize the dosage for the target cells in vivo, andtransformation from varying dosages achieved in culture cells of thesame type as the target cell type can be monitored. Often a single dosecan be sufficient; however, the dose can be repeated if desirable. Thedosage should not be so large as to cause adverse side effects.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can also be adjusted by the individual physician inthe event of any complication.

For administration to a cell in a subject, the composition, once in thesubject, will of course adjust to the subject's body temperature. For exvivo administration, the composition can be administered by any standardmethods that would maintain viability of the cells, such as by adding itto culture medium (appropriate for the target cells) and adding thismedium directly to the cells. As is known in the art, any medium used inthis method can be aqueous and non-toxic so as not to render the cellsnon-viable. In addition, it can contain standard nutrients formaintaining viability of cells, if desired. For in vivo administration,the complex can be added to, for example, a blood sample or a tissuesample from the patient, or to a pharmaceutically acceptable carrier,e.g., saline and buffered saline, and administered by any of severalmeans known in the art. Examples of administration include parenteraladministration, e.g., by intravenous injection including regionalperfusion through a blood vessel supplying the tissues(s) or organ(s)having the target cell(s), or by inhalation of an aerosol, subcutaneousor intramuscular injection, topical administration such as to skinwounds and lesions, direct transfection into, e.g., bone marrow cellsprepared for transplantation and subsequent transplantation into thesubject, and direct transfection into an organ that is subsequentlytransplanted into the subject. Further administration methods includeoral administration, particularly when the composition is encapsulated,or rectal administration, particularly when the composition is insuppository form. A pharmaceutically acceptable carrier includes anymaterial that is not biologically or otherwise undesirable, i.e., thematerial may be administered to an individual along with the selectedcomplex without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical composition in which it is contained.

Specifically, if a particular cell type in vivo is to be targeted, forexample, by regional perfusion of an organ or tumor, cells from thetarget tissue can be biopsied and optimal dosages for import of thecomplex into that tissue can be determined in vitro, as described hereinand as known in the art, to optimize the in vivo dosage, includingconcentration and time length. Alternatively, cultured cells of the samecell type can also be used to optimize the dosage for the target cellsin vivo.

For either ex vivo or in vivo use, the complex can be administered atany effective concentration. An effective concentration is that amountthat results in reduction, inhibition or prevention of the viralinfection or in reduction or inhibition of the transformed phenotype ofthe cells.

A nucleic acid can be administered in any of several means, which can beselected according to the vector utilized, the organ or tissue, if any,to be targeted, and the characteristics of the subject. The nucleicacids, if desired in a pharmaceutically acceptable carrier such asphysiological saline, can be administered systemically, such asintravenously, intraarterially, orally, parenterally, subcutaneously.The nucleic acids can also be administered by direct injection into anorgan or by injection into the blood vessel supplying a target tissue.For an infection of cells of the lungs or trachea, it can beadministered intratracheally. The nucleic acids can additionally beadministered topically, transdermally, etc.

The nucleic acid or protein can be administered in a composition. Forexample, the composition can comprise other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc. Furthermore,the composition can comprise, in addition to the vector, lipids such asliposomes, such as cationic liposomes (e.g., DOTMA, DOPE,DC-cholesterol) or anionic liposomes. Liposomes can further compriseproteins to facilitate targeting a particular cell, if desired.Administration of a composition comprising a vector and a cationicliposome can be administered to the blood afferent to a target organ orinhaled into the respiratory tract to target cells of the respiratorytract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell.Mol. Biol. 1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci USA84:7413-7417 (1987); U.S. Pat. No.4,897,355.

For a viral vector comprising a nucleic acid, the composition cancomprise a pharmaceutically acceptable carrier such as phosphatebuffered saline or saline. The viral vector can be selected according tothe target cell, as known in the art. For example, adenoviral vectors,in particular replication-deficient adenoviral vectors, can be utilizedto target any of a number of cells, because of its broad host range.Many other viral vectors are available, and their target cells areknown.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

EXAMPLES

Selective Elimination of Virally Infected Cells from a Cell Culture

Rat intestinal cell line-1 cells (RIE-1 cells) were standardly grown inDulbecco's modified Eagle's medium, high glucose, supplemented with 10%fetal bovine serum. To begin the experiment, cells persistently infectedwith reovirus were grown to near confluence, then serum was removed fromthe growth medium by removing the medium, washing the cells in PBS, andreturning to the flask medium not supplemented with serum. Typically,the serum content was reduced to 1% or less. The cells are starved forserum for several days, or as long as about a month, to bring them toquiescence or growth arrest. Media containing 10% serum is then added tothe quiescent cells to stimulate growth of the cells. Surviving cellsare found to not be persistently infected cells by immunohistochemicaltechniques used to establish whether cells contain any infectious virus(sensitivity to 1 infectious virus per ml of homogenized cells).

Cellular Genomic DNA Isolation

Gene Trap Libraries: The libraries are generated by infecting the RIE-1cells with a retrovirus vector (U3 gene-trap) at a ratio of less thanone retrovirus for every ten cells. When a U3 gene trap retrovirusintegrates within an actively transcribed gene, the neomycin resistancegene that the U3 gene trap retrovirus encodes is also transcribed, thusconferring resistance to the cell to the antibiotic neomycin. Cells withgene trap events are able to survive exposure to neomycin while cellswithout a gene trap event die. The various cells that survive neomycinselection are then propagated as a library of gene trap events. Suchlibraries can be generated with any retrovirus vector that has theproperties of expressing a reporter gene from a transcriptionally activecellular promoter that tags the gene for later identification.

Reovirus selection: Reovirus infection is typically lethal to RIE-1cells but can result in the development of persistently infected cells.These cells continue to grow while producing infective reovirusparticles. For the identification of gene trap events that conferreovirus resistance to cells, the persistently infected cells must beeliminated or they will be scored as false positives. We have found thatRIE-1 cells persistently infected with reovirus are very poorly tolerantto serum starvation, passaging and plating at low density. Thus, we havedeveloped protocols for the screening of the RIE- 1 gene trap librariesthat select against both reovirus sensitive cells and cells that arepersistently infected with reovirus.

-   1. RIE-1 library cells are grown to near confluence and then the    serum is removed from the media. The cells are starved for serum for    several days to bring them to quiescent or growth arrest.-   2. The library cells are infected with reovirus at a titer of    greater than ten reovirus per cell and the serum starvation is    continued for several more days.-   3. The infected cells are passaged, (a process in which they are    exposed to serum for three to six hours) and then starved for serum    for several more days.-   4. The surviving cells are then allowed to grow in the presence of    serum until visible colonies develop at which point they are cloned    by limiting dilution.-   MEDIA: DULBECCO'S MODIFIED EAGLE'S MEDIUM, HIGH GLUCOSE (DME/HIGH)    Hyclone Laboratories cat. no. SH30003.02.-   NEOMYCIN: The antibiotic used to select against the cells that did    not have a U3 gene trap retrovirus, e.g. GENETICIN, from Sigma.    [cat. no. G9516].-   RAT INTESTINAL CELL LINE-1 CELLS (RIE-1 CELLS): These cells are from    the laboratory of Dr. Ray Dubois (VAMC). They are typically cultured    in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal    calf serum.-   REOVIRUS: Laboratory strains of either serotype 1 or serotype 3 are    used. They were originally obtained from the laboratories of    Bernard N. Fields (deceased). These viruses have been described in    detail.-   RETROVIRUS: The U3 gene trap retrovirus used here were developed by    Dr. Earl Ruley (VAMC) and the libraries were produced using a    general protocol suggested by him.-   SERUM: FETAL BOVINE SERUM Hyclone Laboratories cat. no. A-1115-L.    Identification Tags for Isolated Nucleic Acids

Genomic sequences, tagged with a vector, such as the U3 gene trapvector, are given a number corresponding to the genomic library ofmutant cells from which the sequence was isolated., and a letterindicating a unique member of the library. More than one sequence withthe same number and letter indicates multiple, unique sequences obtainedfrom the genome surrounding the vector insert that “tagged” the gene.Such genomic sequences are obtained using vector-based primers, fromwhich sequencing occurs 3′ to 5′ or 5′ to 3′. In the former case, torecover the orientation of the gene into which the vector inserted, thesequence derived from the vector primer must be reversed andcomplemented. Such reverse complement sequences are designated “rE”. Inthe case of genome sequencing from a primer that occurs 5” to 3′ (i.e.the primer is at the 3′ end of the vector), no changes are needed, sincethe derived sequence is the sequence as it appears in the genedisrupted. Such sequences are designated “B4”. Homologies indicatedbelow each genomic sequence are in the positive direction, unlessexplicitly noted to be on the negative strand. As an example, SEQ ID NO.27 comprises a nucleic acid sequence encoding a novel polypeptide on thepositive strand, while the negative strand encodes ferritin. SEQ ID NO:Lab Designation 1 32-3-2#1E/-rE 2 L191B2E#1-RE 3 L191B2E#3+-rE 421-5-9E-RE homology to: emb/AL021154/HS15005 human DNA sequence 514A14E-rE 6 4cx-b4 7 5a-b4 8 6BSA12-B4 9 X7B/B4 10 x27b4f_1 11 12C#A-rE12 10-3b(5/2/96)/-rE 13 10_4B_4-rE 14 6BE60-rE homology to:alpha-trophomyosin 15 19D3E-rE 16 L19D16E-rE 17 2b_rE 18 14_24_#6-rE 197A7′-rE homology to: annexin II/dynein I 20 L12cx#6-rE homology to: gb:X51760 human zinc finger protein ZFP-36 21 L12cx#11-rE 22 19D5E-rEhomology to: 6-pyruvoyl-tetrahydropterin synthase (gb/M77850/RAT6PTHS)23 12_3b#7-rE 24 12_3B#8-RE homology to: gb/AA871174/vq32a08.r1 Barskadbowel MPLRBg Mus musculus cDNA clone 10959265′ 25 9B27-2-E homology to:RAT LOCUS RNU53922 04-MAY-1996; Rattus norvegicus DnaJ-like protein(RDJ1) mRNA, complete Cds, ACCESSION U53922 (on negative strand) 26x15-rE 27 X11-rE homology to: ferritin H (on the negative strand) 28X20-rE homology to: LOCUS RATGL5A Rat NICER element (GL5-14)5′ longterminal repeat, Acc. No. M59028 M33535N1D 29 X4-rE 30 14A7E-rE homologyto: MMSMAD7 3681 bp mRNA ROD 31-JUL-1998 DEFINITION Mus musculus mRNAfor Mad-related protein Smad7, 149 bases 31 14A13E-rE 32 14_7#2E-rEhomology to: N-acetylglucosaminyltransferase I 33 12CX#6-rE homology to:gb|AA522204|AA522204 vf98g09_r1 Soares mouse mammary gland NbMMG Musmusculus cDNA clone 851872; also 5′ similar to gb X51760 zinc fingerprotein ZFP-36 (HUMAN), gb L20450 Mus musculus DNA-binding protein mRNA,complete cds (MOUSE); Length = 442, 925 bases (shares homology with SEQID NO: 20) 34 12C_2B#9E-rE 35 12CX#11E-rE 36 x5-rE 37 8C5_11-rE 38191E2E-rE 39 19_7AE-rE 40 19_9BE-rE homology to: LOCUS HS347M6 56583 bpDNA PRI 14-JAN- 1998 Human DNA sequence from PAC 347M6 on chromosomeXq22, CSTF2 (Cleavage Stimulation Factor, CF-1, Polyadenylation Factor)64 kD subunit gene 41 191E9E-rE 42 191E8E-rE 43 14C_2E/-rE homology to:gb/H31084/EST104778 Rattus sp. cDNA - 5′ end similar to signalrecognition particle subunit(19 kDa) (on negative strand) 44 14H1E-rE 4514G3E-rE 46 14G_2E-rE 47 6_3_6_2E/-rE homology to: Rattus norvegicuscis-golgi gp130 (on negative strand); and a HUMAN EST (on positivestrand) AI127398; qb70g11.x1 Soares fetal heart NbHH19W Homo sapienscDNA clone (1705508 3′ mRNA sequence) 48 14H4E/-rE 49 18A_8_4E-rE 5018A_8_1E-rE 51 SCB2_19E-rE 52 L197B3E-rE 53 L195C5E-rE homology to: H.pylori and C. jeuni 54 21_5_7E-rE homology to: id3 gene;emb|AL021154|HS150O5 Human DNA sequence from clone 150O5; HTGS phase 1[Homo sapiens]; containing the E2F2 gene for transcription factor E2F-2and the ID3 gene for Inhibitor of DNA binding 3 (dominant negativehelix-loop-helix protein), 1R2, Length = 133667, 971 bases 55 L195B1E-rEhomology to: vK72b07.s1 Knowles Solter mouse 2 cell Mus musculus cDNAclone 960181 5′ 56 L194c4E-rE 57 L193A1E#A-rE 58 L192A3E-rE 59 L1739E-rE60 L192B3E#13-rE contains sequence identical to: insulin growthfactorII/mannose-6-phosphate receptor 61 3 2 4 rE located in the sameregion of the genome as calcyclin, but the gene is “read” in theopposite direction 62 36 7 1 a-rE 63 36 5 1 4 a-rE 64 34 25 5a-rE ratsatellite DNA (RATRSSID 93 bp, ROD 12-MAR-1984) 65 34 24-126/rE homologyto: HSU49928 (3096 bp mRNA) PRI 06-APR-1998, Homo sapiens TAK1 bindingprotein (TAB1) mRNA, complete cds, ACCESSION U49928 NID g1401125, andHS333H23 (142274 bp DNA) HTG 17-JUL-1998 Human DNA sequence 66 3423-1/rE 67 36 5 2-6/rE 68 36 5 2-196/rE 69 34 23-3/rE homology to:gb|L16546|RATAP1X Rat P-glycoprotein (mdr1b) gene 70 34 25 23-rE 71 36 52-196/rE 72 31 3 9/rE homology to: AA798638 568 bp mRNA EST 10-FEB-1998,vw34b06.r1 Soares mouse mammary gland NbMMG Mus musculus cDNAclone1245683 5, mRNA sequence, 824 bases. 73 31 3 6-2-rE 74 31 3 17-rE75 31 3 5-rE homology to: AF046001 2347 bp mRNA PRI 19-FEB-1998, Homosapiens zinc finger transcription factor (ZNF207) mRNA, complete Cds,833 bases. 76 31 3 15#1/rE 77 24 3 5#1/rE 78 31 4 4#1/rE 79 31 3 19#2/rE80 31 4 5#1/rE 81 24 9 3#2/rE 82 L24_26_1-BL homology to: AI045472 396bp mRNA EST 06-JUL-1998, UI-R-C1-jz-h-09-0-UI.s2 UI-R-C1 Rattusnorvegicus cDNA cloneUI-R-C1-jz-h-09-0-UI 3′, mRNA sequence. 83L24_26_1-B4 84 L22_5A1/rE 85 L24_3_2B/rE 86 L24 4-2/rE 87 L24 5-2/rE 88L24 5-3/rE 89 (15-)L28AP/rE 90 L24 26-10/rE homology to: LOCUS R06687403 bp mRNA EST 03-APR-1995; yf10a10.r1 Soares fetal liver spleen 1NFLSHomo sapiens cDNA clone 126426 5′ 91 L24 26-2A/rE 92 L24 26-2B/rEhomology to: gb|AA590026|AA590026 vm22g03.r1 Knowles Solter mouseblastocyst B1 Mus musculus cDNA clone 990964, 459 bases, 139A; andRattus norvegicus Eker rat-associated intracisternal-A particle element93 14 7#2E-rE homology to: N-acetylglucosaminyltransferase I; thissequence shares homology with SEQ ID NO: 32. 94 x18 95 31_3_9-rE 9631_3_6_2-rE 97 31_3_17-rE 98 31_3_15#1-rE 99 24_3_5#1-rE 100 31_4_4#1-rE101 31_3_19#2-rE 102 31_4_5#1-rE 103 24_9_3#2-rE 104 14XD#12E-rE 10570A-rE 106 31-3-4-rE 107 3_6_9-NeoG-rE 108 31_4_2-rE 109 3_2_13-rEhomology to: calcyclin 110 3_2_4-E homology to: pistlre-alpha 1 (withhomology to calcyclin on negative strand) 111 L25-10/-rE homology to:calcyclin 112 L24-4-3/-rE 113 L24-9-1-rE rat id sequence 11417-L25-27#7-rE homology to: calcyclin 115 L21C1E-rE homology to:calcyclin 116 L24-5-3BE-rE. homology to: LOCUS H32572 310 bp mRNA EST08-SEP-1995 EST107805 Rat PC-12 cells, untreated Rattus sp cDNA 5′ end,ACCESSION H32572, and LOCUS AA858747 470 bp mRNA EST 10-MAR-1998UI-R-A0-bb-e-01-0-UI.s1 UI-R-A0 Rattus norvegicus cDNA cloneUI-R-A0-bb-e-01-0-UI, 3′ similar to gb|AA473081|AA473081 vd44b07-r1Barstead MPLRB1 Mus musculus cDNA clone 803413 5′ mRNA sequence 117L24-4-2BE-rE homology to: LOCUS MMU51002 6495 bp DNA ROD 16-JAN- 1997Mus musculus D1x-2 gene, complete cds, ACCESSION U51002 NID g1477589 11817-3-3B-B4 119 L24-26-3/-rE homology to: RNU23776, DNA ROD 10-AUG-1995,Rattus norvegicus Eker rat-associated intracisternal-A particle element120 12_2B#2-rE 121 05-17-3-3He-1-T7 122 21_5_8E-rE homology to:emb|AL021154|HS150O5 Human DNA sequence from clone 150O5; 1p36_13-36_22,contains the E2F2 gene for transcription factor E2F-2 and the ID3 genefor Inhibitor of DNA binding 3(dominant negative helix-loop-helixprotein, 1R2, Length = 133667, 971 bases 123 X18H-t7 124 18A_8_4E-rE 125L24-5-2BE-rE 126 L24-4-2AE-rE 127 L24-10-1BE-rEGenes Necessary for Viral Infection

Some of the isolated sequences dislcosed here comprise sequence encodingthe following proteins: tristetraprolin (human ZFP-36),6-pyruvoyltetrahydropterin synthase, a eukaryotic DnaJ-like protein, ID3(inhibitor of DNA binding 3), N-acetylglucos-aminyltransferase I(mGAT-1), cleavage stimulation factor (CSTF2), TAKI binding protein,human zinc transcription factor ZPF207, Dlx2, Smad7 (Mad-relatedprotein), and P-glycoprotein (mdrlb).

Isolation of Cellular Genes that Suppress a Malignant Phenotype

We have utilized a gene-trap method of selecting cell lines that have atransformed phenotype (are potentially tumor cells) from a population ofcells (RIE-1 parentals) that are not transformed. The parental cellline, RIE-1 cells, does not have the capacity to grow in soft agar or toproduce tumors in mice. Following gene-trapping, cells were screened fortheir capacity to grow in soft agar. These cells were cloned and genomicsequences were obtained 5′ or 3′ of the retrovirus vector, i.e. SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:36 or SEQ ID NO:94; sequences designated with an “x” inthe clone name). All of the cell lines behave as if they are tumor celllines, as they also induce tumors in mice.

Of the cell lines, two are associated with the enhanced expression ofthe prostaglandin synthetase gene II or COX 2. It has been shown thatdisruption of gene function by retroviral targeting of an upstream genehas lead to increased expression of a downstream gene product, COX 2.When a small molecule inhibitor of COX 2 enzyme was added, reversion ofthe transformed phenotype occurred. The COX 2 gene has been found to beincreased in pre-malignant adenomas in humans and overexpressed in humancolon cancer. Inhibitors of COX 2 expression also arrests the growth ofthe tumor. One of the cell lines, x18 (SEQ ID NO:94), has disrupted agene that is now represented in the EST (dbest) database, but the geneis not known (not present in GenBank).

Each of the genes from which the provided nucleotide sequences isisolated (and all clones designated with an “x”) represents a tumorsuppressor gene. The mechanism by which the disrupted genes may suppressa transformed phenotype is at present unknown. However, each onerepresents a tumor suppressor gene that is potentially unique, as noneof the genomic sequences correspond to a known gene. The capacity toselect quickly tumor suppressor genes may provide unique targets in theprocess of treating or preventing (potential for diagnostic testing)cancer.

Isolation of Entire Genomic Genes

An isolated nucleic acid of this invention (whose sequence is set forthin any of SEQ ID NO:1 through SEQ ID NO:127), or a smaller fragmentthereof, is labeled by a detectable label and utilized as a probe toscreen a rat genomic library (lambda phage or yeast artificialchromosome vector library) under high stringency conditions, i.e., highsalt and high temperatures to create hybridization and wash temperature5-20° C. Clones are isolated and sequenced by standard Sangerdideoxynucleotide sequencing methods. Once the entire sequence of thenew clone is determined, it is aligned with the probe sequence and itsorientation relative to the probe sequence determined. A second andthird probe is designed using sequences from either end of the combinedgenomic sequence, respectively. These probes are used to screen thelibrary, isolate new clones, which are sequenced. These sequences arealigned with the previously obtained sequences and new probes designedcorresponding to sequences at either end and the entire process repeateduntil the entire gene is isolated and mapped. When one end of thesequence cannot isolate any new clone, a new library can be screened.The complete sequence includes regulatory regions at the 5′ end and apolyadenylation signal at the 3′ end.

Isolation of cDNAs

An isolated nucleic acid (whose sequence is set forth in any of SEQ IDNO:1 through SEQ ID NO:127), or a smaller fragment thereof, oradditional fragments obtained from the genomic library, that containopen reading frames, is labeled by a detectable label and utilized as aprobe to screen a portions of the present fragments, to screen a CDNAlibrary. A rat CDNA library obtains rat cDNA; a human cDNA libraryobtains a human cDNA. Repeated screens can be utilized as describedabove to obtain the complete coding sequence of the gene from severalclones if necessary. The isolates can then be sequenced to determine thenucleotide sequence by standard means such as dideoxynucleotidesequencing methods.

Serum Survival Factor Isolation and Characterization

The lack of tolerance to serum starvation is due to the acquireddependence of the persistently infected cells for a serum factor(survival factor) that is present in serum. The serum survival factorfor persistently infected cells has a molecular weight between 50 and100 kD and resists inactivation in low pH (pH2) and chloroformextraction. It is inactivated by boiling for 5 minutes [oncefractionated from whole serum (50 to 100 kD fraction)], and in low ionicstrength solution [10 to 50 mM].

The factor was isolated from serum by size fraction using centriprepmolecular cut-off filters with excluding sizes of 30 and 100 kd(Millipore and Amnicon), and dialysis tubing with a molecular exclusionof 50 kd. Polyacrylamide gel electrophoresis and silver staining wasused to determine that all of the resulting material was between 50 and100 kd, confirming the validity of the initial isolation. Furtherpurification was performed on using ion exchange chromatography, andheparin sulfate adsorption columns, followed by HPLC. Activity wasdetermined following adjusting the pH of the serum fraction (30 to 100kd fraction) to different pH conditions using HCl and readjusting the pHto pH 7.4 prior to assessment of biologic activity. Low ionic strengthsensitivity was determined by dialyzing the fraction containing activityinto low ionic strength solution for various lengths of time andreadjusting ionic strength to physiologic conditions prior todetermining biologic activity by dialyzing the fraction against themedia. The biologic activity was maintained in the aqueous solutionfollowing chloroform extraction, indicating the factor is not a lipid.The biologic activity was lost after the 30 to 100 kd fraction wasplaced in a 100° C. water bath for 5 minutes.

Isolated Nucleic Acids

Tagged genomic DNAS isolated were sequenced by standard methods usingSanger dideoxynucleotide sequencing. The sequences were run throughcomputer databanks in a homology search. These genes can be therapytargets particularly because disruption of one or both alleles resultsin a viable cell.

1. An isolated nucleic acid comprising a nucleotide sequence set forthin SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:57,SEQIDNO:58, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ ID NO:80,SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQID NO:108, SEQ ID NO:112, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, and SEQ IDNO:127. 2-5. (canceled)
 6. A host cell containing the nucleic acid ofclaim
 1. 7. A nucleic acid comprising a nucleic acid that selectivelyhybridizes under stringent conditions with the nucleic acid of claim 1.8-13. (canceled)
 14. A polypeptide comprising the amino acid sequenceencoded by the nucleic acid of claims
 1. 15-23. (canceled)
 24. A methodof screening a compound for effectiveness in treating or preventing aviral infection, comprising administering the compound to a cellcontaining a cellular gene comprising the nucleic acid set forth in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82; SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ IDNO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108,SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ IDNO:113, SEQ IDNO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQID NO:118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO: 126, or SEQ IDNO: 127, or a homolog thereof, and functionally encoding a gene productnecessary for reproduction of the virus in the cell but not necessaryfor survival of the cell and detecting the level and/or activity of thegene product produced, a decrease or elimination of the gene productand/or gene product activity indicating a compound effective fortreating or preventing the viral infection. 25-27. (canceled)
 28. Amethod of screening a compound for effectiveness in treating a viralinfection, comprising administering the compound to a cell containing acellular gene functionally encoding a gene product whose overexpressioninhibits reproduction of the virus but does not prevent survival of thecell and detecting the level of the gene product produced, an increasein the gene product indicating a compound effective for treating theviral infection.
 29. (canceled)